Figure 1: The CD82 scaffold regulates PKCα expression and activation.
From: Tetraspanin CD82 Regulates the Spatiotemporal Dynamics of PKCα in Acute Myeloid Leukemia
(A) Cartoon depicting mutated palmitoylation sites within CD82 and mCherry fusion. Flow cytometry analysis of (B) total and (C) surface CD82 expression using CD82KD, control, CD82OE, and Palm-CD82OE KG1a cells (Biolegend, ASL-24). (n ≥ 3 experiments; error bars indicate SD; mean fluorescence intensity normalized to control levels). (D) Western blot analysis for total PKCα (Cell Signaling #2056, polyclonal) and phospho-PKCα (Thr638) expression. Densitometric analysis of (E) total and (F) phosphorylated PKCα expression from Western blot analyses (n ≥ 4 experiments; error bars indicate SD). (G) Real-time PCR analysis of KG1a cells. (H) Immunofluorescence imaging of CD82 (Biolegend, ASL-24) and PKCα-488 (primary, abcam, Y124; secondary, Invitrogen, rabbit-488) under resting and 1 hr of PMA treatment with corresponding line scan plots for both channels. All channels were scaled equally across conditions. (I) Western blot analysis of total and phosphorylated PKCα expression following PMA stimulation (n ≥ 4 experiments; error bars indicate SD). (J) Cells were treated with DMSO, PMA or PMA + MG132 (10 uM) for 4 hrs and total and phospho-PKCα were quantified using Western blot analysis and densitometry. (n ≥ 4 independent experiments; error bars indicate SD; post-hoc unpaired t-test).
